Figure 1. Caspase-1 expression in immune cells. Caspase-1 expression is
shown for (A-C) total (CD3+) T-cells,
CD4+ T cells, non-CD3+ T cell
lymphocytes, and myeloid cells. (D-F) Caspase 1 expression in
CD4+ T cells is correlated with γ/δ T cells, NK cells
(CD3-CD56/16+) and CRTH2+ CD4+ T cells. Individual
patient data are shown and the frequency of caspase-1+
CD4+ T cells are significantly elevated at baseline in
COVID-19 patients (n =29) compared to healthy (n =28)
participants with and without nigericin stimulation. All p-values are by
unpaired and 2-tailed Student’s t test or linear regression
analysis. *p<0.05, ***p<0.001,
****p<0.0001
Figure 2. Caspase-1 expression in CD4+ T cells of
non-COVID-19 patients (unexposed and uninfected adult and pediatric
patients with allergic/immunological disorders). Open symbols are
resting non-stimulated CD4+ T cells. Closed symbols
represent caspase-1 expression in nigericin stimulated
CD4+ T cells. Different symbols represent different
disease states. Adults (>18 years) are represented with an
A (n =65) and pediatric subjects (<18 years) represented
with a P (n =39) in the bottom of the graph. CRS; chronic
rhinosinusitis, CVID; common variable immune deficiency, and CIU;
chronic idiopathic urticaria. The diagnosis and T-cell caspase-1 data
are retrospective data from medical records of patients presenting to an
Allergy Immunology Clinic for an immunological evaluation. Control
patient data was generated during clinical assay validation of T-helper
cell caspase-1 assay (n =45 for adults and n =39 for
pediatrics). All p-values are by unpaired and 2-tailed
Student’s t test. *p<0.05, ***p<0.001,
****p<0.0001.
Figure 3. Caspase-1 expression on CD4+ T cells in
post-COVID-19 Health Care Workers. Blood samples were analyzed at least
90 days after SARS-CoV-2 exposure in healthcare workers. Patients with
no exposure history and negative PCR to SARS-CoV-2 were used as
controls. Solid black circles represent symptomatic, green circles
represent non-symptomatic patients. Exposure indicates being in close
proximity to SARS-CoV-2 infected patients in the absence of personal
protection equipment. Persistent post-COVID19 symptoms correlated with
elevated caspase-1 expression in T-helper cells (p< 0.05).
Figure 4. Effect of caspase inhibition on CD4+ T cells
in COVID-19 patients. Samples from healthy and COVID-19 subjects
incubated with caspase inhibitors: EMR or VX765. Activated caspase-1 was
measured by flow cytometry using a Fam-FLICA probe. Emricasan at 1μM
concentration induces the strongest suppression of
CD4+ T cell caspase-1 in unstimulated cells
(p< 0.01), whereas the selective caspase-1 inhibitor VX-765
does not induce a similar effect. Krustal-Wallis ANOVA test with Tukey
multiple comparisons for >2 group comparisons were used.
For P values are as follows: *p<0.05, **p<0.01.
Experiments represent n =3.
Figure 5. Caspase 3/7 activity in red blood cells (RBC) derived from
COVID-19 patients. Blood samples were analyzed from hospitalized
patients with SARS-CoV-2 infection. A) RBC contamination of the PBMC
layer after Ficoll separation. B) Analysis of caspase 3/7 activity in
COVID-19 patients and healthy controls. Some experiments were done using
plasma from COVID-19 or subjects with influenza with incubated with RBCs
from health uninfected donors as indicated. COVID-19 patients RBCs show
elevated caspase 3/7 (p< 0.01) and EMR has a significant
suppressive activity on this expression (p< 0.05). Plasma from
hospitalized COVID-19 patients induces caspase 3/7 in health RBCs on
overnight incubation (p< 0.01). Krustal-Wallis ANOVA test with
Tukey multiple comparisons for >2 group comparisons were
used. For p values are as
follows: *p<0.05, **p<0.01. Experiments representn =3.
Figure 6. Emricasan mechanism of action.
FIGURE 1